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ON Underwater-Transparent Nanodendritic Coatings for

Directly Monitoring Cancer Cells

Gao Yang , Hongliang Liu , Xueli Liu , Pengchao Zhang , Chao Huang , Tailin Xu , Lei Jiang , and Shutao Wang *

Engineering surface coatings not only possess great signifi -cance in fundamental research to understand complex biolog-ical phenomena, [ 1 ] but also promise a wide range of biological applications, such as artifi cial blood vessels, [ 2 ] bone implants, [ 3 ] and contact lenses. [ 4 ] With the rapid development of nanomate-rials and nanotechnology, to fi ne tune surface nanostructures of surface coating represents an effective strategy to improve their biological functions. [ 5 ] Recently, in the fi eld of rare cell detection, several functionalized nanostructured surfaces, such as silicon nanopillar [ 6 ] and TiO 2 nanofi ber, [ 7 ] have been reported to exhibit the capability of recognizing and capturing cancer cells with high effi ciency. However, those nanostructured coat-ings are limited in monitoring cancer cells because they are not transparent under water, for example, simultaneous bright-fi eld and fl uorescence imaging of captured cancer cells, and real-time observation of the process of detecting cancer cells by microscopy. Therefore, it is an urgent demand to develop underwater-transparent nanostructured coatings that allow directly monitoring cancer cells, without compromise of cell capture effi ciency.

Herein, we demonstrate that an underwater-transparent nanodendritic silica coating, modifi ed with epithelial-cell-adhesion-molecule antibody (anti-EpCAM), exhibits outstanding dual capability for effi ciently capturing and directly monitoring cancer cells from whole blood samples. By a three-step template method, we easily fabricated the unique silica coatings with nan-odendritic structures. The as-prepared coatings exhibited high cell capture effi ciency by enhanced topographic interactions between cancer cells and the unique nanodendritic structure. On the other hand, the underwater transparency of the as-pre-pared nanodendritic coatings allows for directly monitoring cap-tured cells by light microscopy. We believe that this underwater-transparent nanostructured coating represents an example of multifunctional surface coatings for biomedical applications.

We fabricated the nanodendritic silica coating through a three-step template process, as illustrated in Figure 1 a. Taking candle soot as the template provides a robust, inexpensive way developed by Deng et al. to fabricate nanomaterials, for example, an air-transparent superamphiphobic coating. [ 8 ] In our experiments, we fi rst deposited a layer of candle soot on a quartz substrate as the template. The deposited candle soot exhibits a dendritic-like network, consisting of physically con-nected, approximately spherical soot particles with a diameter ranging from 19 to 43 nm (Figure 1 b). [ 9 ] Then, we coated the candle-soot template with a silica shell by chemical vapor depo-sition of silicon tetrachloride. [ 10 ] The thickness of the silica shell is in the range of 23–45 nm (Figure 1 c,d). Finally, we removed the template by calcining the soot/silica core–shell nanostruc-ture at 600 °C for 2 h. After this three-step template process, we obtained a nanodendritic coating with the dendritic feature similar to the candle soot template (Figure 1 d). Moreover, the time of depositing candle soot can effi ciently control the thick-ness of the as-prepared coatings. Therefore, we fabricated a series of relatively uniform nanodendritic coatings with thick-nesses varying from ca. 0.2 to ca. 22.4 μ m (Figure S1, Sup-porting Information).

Next, we determined whether the as-prepared nanodendritic coatings can facilitate capturing cancer cells. In our study, the MCF7 cell line was chose as a model for cell capture. This breast cancer cell line processes an overexpressed membrane antigen termed the epithelial cell adhesion molecule (i.e., EpCAM), and is an EpCAM-positive cell line. [ 6a , 11 ] Based on the anti-EpCAM/EpCAM biological recognition, biotinylated anti-EpCAM was covalently immobilized to the nanoden-dritic coating with a 13.0 ± 0.2 μ m thickness (Figure 1 e and see details in Supporting Information) before the cell cap-ture experiment. [ 6a , 11 ] As a control, a fl at quartz substrate was also modifi ed with anti-EpCAM in parallel. After loaded with a cell suspension (MCF7, 10 5 cells mL −1 ) and incubated for 30 min, more than 70% of MCF7 cells were captured by the anti-EpCAM-modifi ed nanodendritic coating, whereas a very small number of cells were immobilized on the anti-EpCAM-modifi ed fl at quartz substrate (Figure S4, Supporting Informa-tion), revealing the superiority of the nanodendritic structure for cell capture. To explore the enhanced cell-affi nity of the anti-EpCAM-modifi ed nanodendritic coating, we further char-acterized the morphologies of captured MCF7 cells by using environmental scanning electronic microscopy (ESEM). The cells protruded large lamellipodia and numerous fi lopodia on the anti-EpCAM-modifi ed nanodendritic coating ( Figure 2 a), an indication of the enhanced adhesion of MCF7 cells with the antibody-coated nanostructures, [ 12 ] whereas the cells exhibited DOI: 10.1002/adhm.201300233

Dr. G. Yang, Dr. H. L. Liu, Dr. X. L. Liu, Dr. P. C. Zhang, Dr. C. Huang, Dr. T. L. Xu, Prof. L. Jiang, Prof. S. T. WangBeijing National Laboratory for Molecular Sciences (BNLMS) Key Laboratory of Organic SolidsInstitute of Chemistry Chinese Academy of Sciences Beijing , 100190 , P. R. China E-mail: stwang@iccas.ac.cn Dr. G. Yang, Dr. X. L. Liu, Dr. P. C. ZhangUniversity of Chinese Academy of Sciences Beijing , 100049 , P. R. China

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fewer fi lopodia on the anti-EpCAM-modifi ed fl at quartz sub-strate (Figure 2 b). This difference suggests that enhanced local topographic interactions may exist between the antibody-coated nanodendritic structures and nanoscaled components of the cellular surface (e.g., fi lopodia and lamellipodia). [ 6a , 13 , 14 ] These results verify that the anti-EpCAM-modifi ed nanodendritic coating is capable of capturing MCF7 cells with high effi ciency.

To optimize the thickness of the coating for cell capture, we performed a series of cell capture experiments on the anti-EpCAM-modifi ed nanodendritic coatings with varied thick-nesses from ca. 0.2 to ca. 22.4 μ m (Figure 2 c). For the target MCF7 cells, the effi ciency of capture increased signifi cantly with the thickness below 5.3 ± 0.1 μ m, presumably due to the enhanced topographic interaction, [ 6a , 13 , 14 ] and reached the max-imum of 73% ± 3% for the 5.3 ± 0.1 μ m thickness. This capture effi ciency is about 18% higher than that of silicon nanopillar in our previous work, [ 6a ] exhibiting the excellent cell capture performance of the coating. To examine specifi city—the essen-tial requirement for rare cell detection—we further conducted a series of cell capture experiments on three EpCAM-negative

cancer cell lines, including Hela cervical cancer cells, rep-resenting adherent cell type; Daudi cells and Jurkat T cells, representing suspension cell type. [ 15 ] For each cell lines, rela-titively low cell numbers were nonspecifi cally adhered on the anti-EpCAM-modifi ed nanodendritic coatings, compared with the target MCF7 cells. The high effi ciency and specifi city of the anti-EpCAM-modifi ed nanodendritic coating with the thickness of 5.3 ± 0.1 μ m leads us to select this optimal coating for sub-sequent studies.

To determine the minimum time required for optimal cell capture, we carried out a series of cell capture experiments at different capture time, using the optimal coating (i.e., the 5.3 ± 0.1 μ m-thickness anti-EpCAM-modifi ed nanodendritic coating) (Figure 2 d). For MCF7 cells, the effi ciency of capture increased dramatically with time increased, probably owing to enhanced topographic interaction, consistent with our previous results, [ 6a , 13 ] and was 73% ± 3% at 30 min. The effi ciency of capture was not enhanced at capture time more than 30 min ( P < 0.05), leading us to ascertain 30 min to be the minimum time for optimal capture. For EpCAM-negative cancer cells

Figure 1. The fabrication of the nanodendritic coating. a) Schematic procedure for the fabrication of the nanodendritic coating. b) Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) (insets) images of a layer of candle soot. c) SEM and TEM (insets) images of the soot/silica core–shell nanostructure. d) SEM and TEM (insets) images of the nanodendritic coating after the candle soot was removed by calcining the soot/silica core–shell nanostructure at 600 °C for 2 h. The scale bar in the inside fi gure of (b,c,d) is 50 nm. e) Schematic procedure for the preparation of the anti-EpCAM-modifi ed nanodendritic coating for cancer cell capture.

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(i.e., Hela, Daudi, and Jurkat T), the nonspecifi c adhesion is quite low and nearly no increment with time increased, an indi-cation of high specifi city of the anti-EpCAM-modifi ed nanoden-dritic coating. In comparison, we also conducted cell capture experiments on the anti-EpCAM-modifi ed fl at quartz substrate (Figure S3, Supporting Information). For target MCF7 cells, there is a very slow increment with time increased, resulting in a quite low effi ciency of about 9% at 30 min. These results demonstrate that the anti-EpCAM-modifi ed nanodendritic coating can capture target cancer cells with high effi ciency and high specifi city at the minimum time of 30 min.

On the other hand, we studied the optical properties of the nanodendritic coatings with varied thicknesses from ≈1.9 to ≈22.4 μ m by the light transmission spectra (Figure S2, Supporting Information). In air, light transmission of these nanodendritic coatings was less than 46% at the wavelength of 500 nm, and decreased signifi cantly with the thickness increased (Figure S2a, Supporting Information), due to the increased light scattering. [ 16 ] Amazingly, when these coat-ings were placed in water, they became highly transparent. As shown in Figure S2 (Supporting Information), their light transmission decreased slightly with the increasing of the thickness, resulting in more than 60% of light transmission over the visible-light range for the thickness less than 13.0 ± 0.2 μ m. Typically, the average optical transmission was up to 81% over the visible-light range for the coating with a thick-ness of 5.3 ± 0.1 μ m (as described above, this coating is optimal

for cell capture after coated with anti-EpCAM). The photo-graphs of Figure 3 a,b explicitly demonstrate the comparison of optical transparency of the nanodendritic coating between in air and under water. It is observed that only the nanoden-dritic coating was placed in water can the letters be easily seen through the coating. In general, nanostructured surfaces are always not transparent because of the inevitable light scattering that reduces light transmission. [ 16 ] However, we demonstrate that our engineered nanodendritic silica coatings exhibit high underwater transparency over the visible-light range. This phenomenon can be ascribed to two reasons. First, the light absorption of the as-prepared silica coating is almost negligible over the visible-light range. [ 17 ] Second, light scattering through the coating is largely reduced by the match of refl ective index between silica ( n ≈ 1.46) and water ( n ≈ 1.33). [ 18 ] These optical studies demonstrate that the nanodendritic coatings is highly transparent under water, owing to the match of refl ective index between silica and water that vastly reduces light scattering.

To demonstrate the unique capability of the underwater-transparent nanodendritic coating for directly monitoring cap-tured cells, we performed optical imaging of MCF7 cells that captured on the optimal coating (i.e., the 5.3 ± 0.1 μ m-thickness anti-EpCAM-modifi ed nanodendritic coating). After stained with DAPI, the morphology of the captured cells’ nuclei was observed to be round under fl uorescence microscope (Figure 3 c). Simul-taneously, using bright-fi eld microscopy, we detected the mor-phology of the cells (Figure 3 d). A typical cell exhibited a round

Figure 2. Quantitative evaluation of cell capture performance of the anti-EpCAM-modifi ed nanodendritic coating. ESEM images of the MCF7 cell captured on a) the anti-EpCAM-modifi ed nanodendritic coating with the thickness of 13.0 ± 0.2 μ m and b) anti-EpCAM-modifi ed fl at quartz substrate. c) Capture effi ciency of different cell lines (MCF7, EpCAM-positive cell line; Hela, Daudi and Jurkat T, EpCAM-negative cell lines) on anti-EpCAM-modifi ed nanodendritic coatings with varied thicknesses from ca. 0.2 to ca. 22.4 μ m for 30 min. d) Capture effi ciency of different cell lines (MCF7, Hela, Daudi and Jurkat T) on the anti-EpCAM-modifi ed nanodendritic coating with the thickness of 5.3 ± 0.1 μ m at different capture time from 5 to 90 min. The error bars represent standard error of the mean of three replicate experiments. Data points labeled with * have a statistically different cell capture with P < 0.05.

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shape in a diameter of ≈20 μ m as marked in the red box. The corresponding merged image showed the distribution of the nuclei (Figure 3 e). These studies demonstrate that the captured cells can be monitored by simultaneous fl uorescence and bright-fi eld imaging to gain combined insights into cytomorphologic features. [ 19 ] In addition, under bright-fi eld microscope, we can directly count the number of captured cells, and further eval-uate the cell capture performance of the anti-EpCAM-modifi ed nanodendritic coating. Moreover, F-actin of the captured MCF7 cells were monitored by CLSM and DIC microscopy. [ 20 ] The MCF7 cell was imaged at different focal depths (Figure 3 f,g). As labeled with rhodamine-phalloidin, the F-actin of the cell was visualized, displaying a circular-like shape by CLSM (top in Figure 3 f). Directly, the same cell was imaged by DIC (middle in Figure 3 f). The corresponding merged images revealed that the

circular-like F-actin was projected on the edge of the cell (bottom in Figure 3 f). These results demonstrate that the underwater-transparent nanodendritic coating is unique in its capability for directly monitoring captured cancer cells.

To explore the potential clinical application of the anti-EpCAM-modifi ed nanodendritic coating, we performed a series of experiments to capture variable rare cancer cells from whole blood samples. Red-dye-stained MCF7 cells were spiked into rat whole blood at concentrations of approximately 20, 50, 100, and 250 cells mL −1 . Under the optimal conditions (i.e., the 5.3 ± 0.1 μ m-thickness anti-EpCAM-modifi ed nano-dendritic coating and 30 min incubation time), a considerable capture yield of 43%–60% of spiked cells was achieved from the whole blood ( Figure 4 a and Table S1, Supporting Information). These result shows vastly improved capture yields compared

Figure 3. Directly monitoring the MCF7 cells that captured on the anti-EpCAM-modifi ed nanodendritic coating with the thickness of 5.3 ± 0.1 μ m. The typical photographs of the nanodendritic coating in a) air and b) under water. c) Fluorescence, d) bright-fi eld, and e) the corresponding merged images of captured cells. The cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) dihydrochloride for DNA content. f) Cross-section images of a captured cell with different focal depth along the Z axis. The cell was stained with rhodamine-phalloidin for F-actin. Confocal laser scanning micros-copy (CLSM) (top), differential interference contrast microscopy (DIC) (middle), the corresponding merged (bottom) images of the cell. Scale bar in (f) is 10 μ m. e) Side-view CLSM of the same MCF7 cell. The position of each image with different focal depth along the Z axis in (f) is demonstrated in the side view of the MCF7 in (g).

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with those observed for the commercialized technology. [ 21 ] As for an artifi cial patient blood samples by spiking nonprestained MCF7 cells, we also can identify cancer cells from nonspecifi -cally adhered white blood cells (WBCs) by a typical three-color immunostaining method. [ 6a , 11a ] As shown in Figure 4 b, DAPI was used to stain DNA content, PE-labeled anti-CK marked the Cytokeratin (a protein marker for epithelial cells) and FITC-labeled anti-CD45 was applied as a marker of WBCs. After image acquisition, the combined information was utilized to distinguish spiked MCF7 cells (CK+/CD45-/DAPI+, 10 μ m < cell sizes < 40 μ m) from WBCs (CK-/CD45+/DAPI+, sizes < 15 μ m) and cellular debris. These results indicate that the anti-EpCAM-modofi ed nanodendritic coatings are capable of effi -ciently capturing rare number of cancer cells from whole blood.

In summary, an underwater-transparent nanodendritic silica coating was fabricated and exhibited unique dual-capability to effi ciently capture and directly monitor cancer cells. The match of refl ective index between silica and water can vastly reduce the light scattering of nanodendritic coating and thus leads to high transparency. The underwater transparency enabled the anti-EpCAM-modifi ed nanodendritic coatings to directly

monitor the captured cancer cells. Moreover, the anti-EpCAM-modifi ed nanodendritic coatings can effi ciently capture rare number of cancer cells from spiked whole blood samples. We believe that this kind of underwater-transparent nanondendritic coatings will provide promising prospects in cell-based cancer research, such as rare cancer cell detection, [ 6a , 22 ] and anticancer drug screen. [ 23 ] The work presented here will offer new clues in the creation of multifunctional nanostructured coatings for biomedical engineering.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements This work was supported by the National Research Fund for Fundamental Key Projects (2012CB933800, 2011CB935700, 2012CB933200), National Natural Science Foundation (21175140, 20974113, 21121001), and the Key Research Program of the Chinese Academy of Sciences (KJZD-EW-M01).

Received: June 11, 2013 Published online: August 15, 2013

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